Abstract LT006: PAX3::FOXO1-targeting PROTACs in fusion-positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma. Fusion-positive (FP) RMS typically results in lower survival rates compared to fusion-negative RMS. FP-RMS is characterized by the presence of tumor-specific fusion oncogenes, most commonly PAX3::FOXO1 or PAX7::FOXO1, created by chromosomal translocations joining the DNA binding domains of PAX3 or PAX7 with the transcriptional activation domain of FOXO1. PAX3::FOXO1 plays a critical role in FP-RMS oncogenesis in both tumor initiation and maintenance. Thus, PAX3::FOXO1 represents an excellent target for therapeutic intervention in FP-RMS for which no new treatment option has been introduced in over 40 years. Following our discovery of small molecules that can directly bind to the fusion protein, we designed PAX3::FOXO1-targeting Proteolysis Targeting Chimeras (PROTACs) using the E3 ligase cereblon (CRBN) ligand via different linkers. This first generation of 15 PAX3::FOXO1-PROTACs demonstrated that endogenous PAX3::FOXO1 can be degraded in FP-RMS cell lines with protein degradation reaching over 50%. To further optimize our PROTACs, we identified SKP1 as the E3 ligase with the highest expression in both FP-RMS patient tumors and cell lines and as a dependency in FP-RMS cell lines which would limit emerging resistance towards SKP1-based PROTACs in contrast to CRBN-based PROTACs. The newly designed SKP1-based PAX3::FOXO1-PROTACs and their CRBN-based counterparts demonstrated on-mechanism PAX3::FOXO1 protein degradation via the proteasome as PAX3::FOXO1 protein levels could be rescued with proteasome inhibitor MG132 and NEDD8-activating enzyme inhibitor MLN4924. Moreover, we confirmed PROTACs-mediated ubiquitination of PAX3::FOXO1 protein. As expected with the loss of PAX3::FOXO1 function, we observe signs of myodifferentiation in FP-RMS cells shown in a spindle-like cell morphology and decreased expression of PAX3::FOXO1 target genes such as MYOD1 and B7-H3. Importantly, treatment of FP-RMS cells with PAX3::FOXO1-PROTACs resulted in impairment in anchorage-independent growth in soft agar which was not observed in non-FP-RMS cells. Taken together, we demonstrate proof of principle of PROTACs targeting the oncogenic fusion protein PAX3::FOXO1. Our designed PROTACs will not only be useful tools in studying PAX3::FOXO1 biology but could also serve as scaffolds for designing clinical-grade molecules to assess the therapeutic potential of PAX3::FOXO1-targeting PROTACs. Citation Format: Nikola Knoll, Kayra Somay, Foram Shah, Purushottam Tiwari, Isabel Frye, Jeffrey Toretsky, Aykut Üren. PAX3::FOXO1-targeting PROTACs in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr LT006.